The capacity of blood to clot and stop flowing from a wound is dependent on the proper functional interaction of a large number of factors and cofactors in the blood coagulation cascade. The ability of clinical laboratories to reliably and conveniently assay for these factors and cofactors in plasma samples from donor patients can be critical in monitoring individuals for whom either inappropriate coagulation episodes (such as occurs, for example, in disseminated intravascular coagulation disease) or inappropriate failure of blood to clot (such as occurs, for example, in hemophilia) is a daily life-threatening problem.
The physiologic basis for these clinically important coagulation problems can be exceedingly complex to diagnose. In most cases, however, diagnosis can be made by detecting either quantitative or functional changes in certain of the biological factors and cofactors which activate or regulate the coagulation process. A number of diagnostic assays have been developed which reliably measure many of the regulatory proteins involved in the coagulation cascade (see, for example, the textbooks by R. Hoffman et al., Hematology: Basic Principles and Practice, New York: Churchill Livingston Inc., 1991; and by C. Kjeldsberg et al., Practical Diagnosis of Hematologic Disorders, Chicago: American Society of Clinical Pathologists Press, 1989). These include such routine coagulation assays as the activated partial thromboplastin time (APTT) assay, the prothrombin time (PT) assay, and the thrombin clotting time (TCT) assay. In spite of extensive research, none of these assays directly measure, in a one-stage assay, the functional status in plasma of the vitamin K-dependent coagulation-inhibiting proteins, protein C and protein S. Because protein C and protein S are both essential in the normal regulatory process of down-regulating the blood coagulation cascade, the lack of any one-stage assay which directly determines both of their functional activities remains a serious deficiency in the art.
The need for a convenient one-stage assay for determining protein C and protein S function in plasma is emphasized by the fact that abnormalities in protein C and protein S function are more common in the population than is usually believed. For example, from 4-to-5% of the population as a whole have genetically-acquired protein C or protein S deficiencies, and a large number of these individuals have a clinical history of recurrent thrombotic (microvascular clotting) episodes (Gladson et al., Thromb. Haemostas. 59: 18, 1988). It is these individuals who should be regularly monitored for the functional activities of these coagulation-inhibiting proteins individuals who are known to be at high risk for recurrent thrombosis (which can be lethal) should be kept on lifelong maintenance anticoagulant therapy, with regular monitoring.
The coagulation enzyme system
To better appreciate the importance of such assays, and how activated protein C and protein S are involved in regulating the blood coagulation cascade, the following brief description of the coagulation enzyme system is provided.
The blood clotting system may best be viewed as a chain reaction involving the sequential activation of inactive enzyme precursors (zymogens) into active serine proteases. These activation events, which take place on the surfaces of cells such as platelets, white blood cells, and endothelial cells, have been divided into two distinct pathways termed the extrinsic and the intrinsic pathways of coagulation. A clot is generated in the intrinsic pathway by activation of those coagulation components which are all contained in (or are "intrinsic to") whole blood. In the extrinsic pathway, components intrinsic to whole blood are reguired along with an externally-supplied coagulation-activating substance known as "tissue factor" (also sometimes referred to as thromboplastin, thrombokinase, or blood coagulation factor III). Tissue factor is a cell surface protein extrinsic to blood, and is expressed by cellular injury. Whether a particular coagulation factor becomes activated in the extrinsic or the intrinsic pathway is important in selecting a particular coagulometric assay with which to detect and evaluate that particular factor.
Thrombin. The multistep coagulation chain reaction uitimately produces the enzyme thrombin, the last serine protease in the coagulation cascade, which, through limited proteolysis, converts fibrinogen molecules into an insoluble gel of fibrin fibers which forms the physical clot. Two key events in the coagulation cascade are the conversion of clotting factor X into an activated form, factor Xa, and the subsequent conversion of prothrombin by factor Xa into thrombin. Both of these conversion reactions occur on cell surfaces, such as, for example, the surfaces of platelets, and both reactions require cofactors. These cofactors, factors V and VIII, are in circulation in the form of relatively inactive precursor molecules. When the first few molecules of thrombin are formed, thrombin loops back and activates factors V and VIII through limited proteolysis. The activated factors, Va and VIIIa, accelerate both the conversion of prothrombin into thrombin and also the conversion of factor X to factor Xa, speeding the clotting process by approximately 100,000 fold. This significant amplification is essential in the timely formation of a clot. As will be discussed below, factors Va and VIIIa are the principal protein targets of activated protein C and protein S.
Thrombomodulin. Some of the thrombin formed during the formation of a clot is also bound by thrombomodulin, an essential reagent used in soluble form in the present invention. Thrombomodulin is a glycoprotein normally found fixed in the surface membranes of endothelial cells that form the lining of all blood vessels. While vascular endothelium once was thought to be a passive barrier which simply channeled the blood, it now is known that endothelial cells are actively involved in the regulation of intravascular coagulation mechanisms, and that much of this activity is due to the thrombomodulin in the endothelial cell membranes.
Thrombin-thrombomodulin complex. Thrombomodulin forms a tight, stoichiometric complex with thrombin, altering both the physical shape and functional properties of thrombin so that it no longer has procoagulant activity, i.e., it no longer converts fibrinogen to fibrin, activates platelets, or converts clotting factors V and VIII to their activated counterparts Va and VIIIa. Rather, the thrombin which is bound by thrombomodulin becomes an efficient activator of protein C. The rate constant for the activation of protein C by thrombin bound in a thrombomodulin-thrombin complex is about 20,000 fold higher than the rate constant for activation of protein C by thrombin found free in solution. Additionally, activation of protein C by thrombin in the absence of thrombomodulin is inhibited by calcium ions, whereas activation by the thrombin/thrombomodulin complex is a calcium-dependent reaction.
Thus, the coagulation-inhibiting effects of thrombomodulin are of two different types. One is a heparin-like anticoagulant effect in which the binding of thrombin to thrombomodulin inhibits the capacity of thrombin to enzymatically convert fibrinogen into fibrin fibers; the anticoagulant heparin exerts a similar antithrombin effect. This is the thrombomodulin-mediated anticoagulant effect most commonly observed by others (for example, see Gomi et al., Blood 75: 1396, 1990).
The second anticoagulant effect of thrombomodulin results from the activation of protein C by the thrombin-thrombomodulin complex. This effect is the key component of the present invention. When activated, protein C has the capacity to enzymatically cleave (via hydrolysis) both factor Va and factor VIIIa, substantially inhibiting both of their clot-promoting activities. In the presence of its vitamin K-dependent cofactor protein S, the rate of activated protein C-mediated hydrolysis of factors Va and VIIIa is increased by about 25 fold compared to hydrolysis in the absence of protein S. Thus, one of the important mechanisms operating in the vascular endothelium to maintain the normal anticoagulant state of the endothelial surface is the pathway by which a thrombin-thrombomodulin complex activates protein C to form a coagulation-inhibiting activated protein C-protein S complex on a cell surface.
The vitamin K-dependency of the protein C and protein S coagulation-inhibiting proteins derives from the fact that a vitamin K-dependent microsomal carboxylase enzyme in the liver forms an unusual amino acid, gammacarboxyglutamic acid, in a post-ribosomal carboxylation step in the precursor proteins of both protein C and protein S. The appearance of the gammacarboxyglutamic acid moieties in these protein molecules is crucial in that it facilitates their efficient binding via a calcium-mediated bridge, to phospholipid-containing surfaces such as, for example, the surfaces of platelets, endothelial cells, and, importantly for in vitro coagulometric assays, phospholipid micelles in solution. As noted below, the binding of the carboxylated glycoproteins to a phospholipid surface allows the proteins to concentrate, interface and interact with one another more efficiently in three-dimensional space. Several in vitro clotting assays take advantage of this fact, in that phospholipids are included among the necessary reagents used in the coagulometric assays.
Phospholipid. All clotting reactions with meaningful rates are viewed as occurring on a surface. Crucial to the efficient interaction of thrombomodulin (the cofactor), thrombin (the enzyme), and protein C (the substrate) is the presence of calcium ions and an integrating surface such as is found, for example, on a soluble phospholipid micelle (a submicroscopic phospholipid sphere). On such a surfacer the three reactants are brought into close proximity, thereby substantially increasing their effective concentrations and their reaction rate many fold. It is believed that all relevant soluble complexes which contain soluble thrombomodulin, thrombin and activated protein C, and which produce vitamin K-dependent anticoagulant activity, also contain an essential surface component. The in vitro clotting assays of the present invention take advantage of this fact by the use of a suitable phospholipid (cephalin) in a preferred embodiment.
Protein C and Protein S assays
Until the present invention, it has not been possible to conveniently measure the functional activities of both protein C and protein S in a simple and rapid one-stage in vitro assay. This is because soluble thrombomodulin has never before been added directly into laboratory coagulometric assays to detect protein C zymogen. Rather, thrombomodulin has been added with thrombin as a preactivation reagent, either as a soluble complex, or in an immobilized form and in conjunction with a process which required either prior separation of protein C from other plasma components or the subsequent removal of activated protein C from other plasma components in order to carry out the final determination of activated protein C in a separate coagulometric or amidolytic assay. Thus, the anticoagulatory effect of soluble thrombomodulin, in the form of soluble thrombomodulin-thrombin complexes, has never been exploited directly in conventional clinical assays.
Protein C assays
For protein C, a number of different assays are available for its determination (as reviewed in detail by Lobermann et al., Behring Inst. Mitt. 79:112, 1986; Vigano-D'Angelo et al., in Biotechnology in Clinical Medicine [Albertini et al., editors] New York: Raven Press, 1989: and more recently by Preissner, Clin. Sci. 78: 351, 1990). The assays generally fall into three categories: antigenic detection assays, chromogenic (amidolytic) assays, and functional coagulometric assays.
Protein C antigenic assays. For determination of protein C antigen concentration in plasma, detection assays include electroimmunoassay, radioimmunoassay, and ELISA-type assays. Not only polyclonal antibodies from rabbit or goat, but also monoclonal antibodies have been used as detecting reagents in these assays. While in vitro detection of protein C with antibodies is a sensitive procedure, antigenic detection provides little or no information regarding the functional capacity of the protein C detected.
Protein C chromogenic assays. Alternatively, because activated protein C is an enzyme, its presence in a plasma sample can be quantified with a chromogenic substrate. Prior activation of protein C, with exposure of the enzymatic active site, is required for expression of its enzymatic activity. However, the relatively small size of the synthetic chromogenic substrate (about 600 daltons) commonly used in these assays may permit satisfactory proteolysis of the synthetic substrate, while missing defects in the functional integrity of protein C which would prevent proteolytic cleavage of factor Va and VIIIa, the biological substrates of activated protein C which each have molecular weights of about 300,000 daltons. Moreover, the interaction of activated protein C with its cofactors (protein S, calcium ions, and phospholipid), which requires that all functional features of the protein C molecule be intact, is not evaluated by chromogenic assays. It is important to be aware of this, since protein C molecules detected in chromogenic assays can still be deficient in functional anticoagulant capacity.
Protein C coagulometric assays. For determination of functional capacity of protein C molecules in a plasma sample, coagulometric assays offer the advantage of evaluating the coagulation-inhibiting activity of activated protein C in the presence of its usual biological substrates and cofactors, thereby reflecting more accurately the physiologic state of the protein C in the particular plasma sample being evaluated.
The protein C in a plasma sample is commonly activated before it is assayed. This activation is done either with or without prior separation of protein C from other plasma components. Isolation of the protein C from the test plasma sample has (up until the present invention) been necessary if the protein C is to be activated by thrombin or by the physiological activator, which is the thrombin/thrombomodulin complex. Unfortunately, because of poor reaction kinetics, activation of protein C with thrombin in the absence of thrombomodulin does not lead to complete activation of protein C in the sample. In contrast, when thrombin is bound to thrombomodulin, the rate of activation is increased by about 20,000 fold over the rate obtained with thrombin alone.
Activation can also be carried out without prior isolation of protein C from the test plasma sample by use of a snake venom activator such as, for example, southern copperhead venom, or the "PROTAC.RTM. C" reagent (the latter from Pentapharm, Basel, Switzerland).
After either of the above procedures, the anticoagulant activity of activated protein C is assessed most commonly either by prolongation of the otherwise routine activated partial thromboplastin time (APTT) assay, or in a factor Xa one-stage coagulometric assay. The clot time in this latter assay is a function of the conversion of prothrombin to thrombin, and is initiated by the addition of exogenous factor Xa to the plasma sample in the presence of calcium ions and a phospholipid component. The assay is sensitive to activated factor V, but not to activated factor VIII. In this system, addition of preactivated protein C from the test plasma sample to a control plasma sample prior to the addition of factor Xa prolongs the clot time as a result of its ability to inactivate factor Va generated during the reaction.
In stark contrast to the commonly used assays discussed above, the novel assay of the present invention does not require preliminary isolation of protein C from a plasma test sample; it permits the activation of protein C with its physiological activator, the thrombin/thrombomodulin complex; and it results in a determination of protein C functional activity in the test sample, all in a simple one-stage procedure.
Protein S assays
Laboratory evaluation of protein S status is complicated by the fact that two forms of protein S are present in plasma. In plasma, about 40% to 50% of the protein S is free and serves as the cofactor for activated protein C. The remaining 50% to 60% of plasma protein S is complexed to C4b binding protein ("C4bBP") and, as such, is unavailable as an anticoagulant. That C4bBP is an acute phase protein and is elevated during inflammation further complicates evaluation of protein S status. This rise in C4bBP favors a transient shift of the protein S to the complexed form, thereby inducing a relative and transient protein S deficiency state. Because protein S which is bound to C4bBP in plasma is no longer able to function as a cofactor with activated protein C, individuals with serious inflammatory disorders are often at risk for thrombosis. This is an acquired situation which will rise and fall, and even disappear, depending on the state of the inflammatory disorder.
In the clinical laboratory, a very limited number of tests are available to assess protein S status, and these are generally antigen detection assays. These tests include polyethylene glycol precipitation of bound protein S with subsequent measurement of the free protein S remaining in the plasma; crossed immunoelectrophoresis for protein S, which separates free and bound forms of protein S but is not quantitative; and enzyme-linked immunosorbent assays (ELISA). ELISA assays are suitable in combination with PEG precipitation to quantitate free protein S. However, while ELISA assays may detect protein S with sensitivity, such antigen detecting assays provide little or no information regarding the functional capacity of the protein S detected. Unfortunately, standardized functional assays for protein S are not yet available in the general clinical laboratory.
It is, therefore, a principal object of the present invention to provide a convenient and reliable one-stage assay by which both of the vitamin K-dependent coagulation-inhibiting proteins, protein C and protein S, can be quantitatively and functionally determined in a blood plasma test sample in a clinical laboratory.
It is another object of the present invention to provide a soluble coagulation-inhibiting complex comprising soluble thrombomodulin, thrombin and protein C, bound to a soluble phospholipid surface. Such a phospholipid surface is found, for example, on soluble phospholipid micelles.
It is still another object of the present invention to provide a soluble complex comprising activated protein C and its cofactor, protein S, bound to a soluble phospholipid surface.
Yet another object is to provide a soluble complex comprising activated protein C, protein S and factor Va bound to a soluble phospholipid surface.
Still another object is to provide a soluble complex comprising activated protein C, protein S and factor VIIIa bound to a soluble phospholipid surface.